In addition to being nutrients that are essential for growth of vertebrates, including humans, polyunsaturated fatty acids in recent years have been frequently reported to be involved in cardiovascular diseases and inflammatory diseases. There have been many reports of findings that the intake of n-3 type polyunsaturated fatty acids, such as docosahexaenoic acid and eicosapentaenoic acid, is particularly useful for human health. There have been reports that the ratio of intake amount of n-3 type polyunsaturated fatty acid to intake amount of n-6 type polyunsaturated fatty acid is important. The industrialized world is characterized by a trend of decreased intake of n-3 type polyunsaturated fatty acids and a trend of increased intake of calories, saturated fatty acids, and n-6 type polyunsaturated fatty acids. This trend is considered to be related to various types of lifestyle-related diseases.
Fish oil is an oil that is rich in n-3 type polyunsaturated fatty acids. The intake of such fish oil is widely recommended, and methods are being devised for the concentration of the n-3 type polyunsaturated fatty acids in fish oil for more efficient intake of n-3 type polyunsaturated fatty acids. The concentration of polyunsaturated fatty acids using lipase reactions is one such method.
Lipases are enzymes that catalyze hydrolysis reactions to decompose oils into free fatty acids and glycerin. Various types of animals and plants and microorganisms are known to have lipases. A given type of lipase does not act similarly for all fatty acids, and the reactivity of a given lipase will vary according to bond position in the glyceride, carbon chain length of the fatty acid, number of double bonds, or the like. It is thus possible to selectively hydrolyze fatty acids using such lipases, and as a result, it becomes possible to concentrate a specific fatty acid within the glyceride fraction. For example, when a lipase produced by a kind of the genus Candida is used, it is known that hydrolysis reaction of fish oil results in concentration of polyunsaturated fatty acids, such as docosahexaenoic acid or the like, in the undecomposed glyceride fraction (Patent Document 1).
Hydrolysis reaction by lipase in this manner is a method that is effective for the concentration of polyunsaturated fatty acids. The concentration of polyunsaturated fatty acid in the glyceride fraction increases with progress of hydrolysis with respect to fatty acids other than those of the target polyunsaturated fatty acid. However, as the concentration in the glyceride fraction of the polyunsaturated fatty acid increases, the hydrolysis reaction slows down in practice, and excess enzyme must be added in order that the hydrolysis reaction proceed further. Although the hydrolysis reaction proceeds further when an excess of enzyme has been added, such excess addition then results in a lowering of yield due to hydrolysis reaction of the target polyunsaturated fatty acid and a lowering of the concentration effect as hydrolysis increases. Moreover, lipase gradually loses activity with the passage of hydrolysis reaction time. It is thus possible to further promote hydrolysis by removing the deactivated enzyme and repeating the reaction using fresh enzyme. However, even in this case, there is a marked decline of yield when the degree of hydrolysis is excessive, and the concentration effect for the target polyunsaturated fatty acid is lost.
When Candida cylindracea-derived lipase is used for hydrolysis of fish oil, it is possible to increase the acid value of the hydrolyzed oil by increasing the utilized amount of lipase, by prolonging the reaction time, or by repeatedly performing hydrolysis using lipase. However, it is reported that although concentration of the target polyunsaturated fatty acid proceeds, when the acid value exceeds roughly 160, the degree of concentration of polyunsaturated fatty acid conversely decreases (Patent Document 1). That is to say, this means that the frequency of hydrolysis of the target polyunsaturated fatty acid becomes higher when the concentration of the target polyunsaturated fatty acid is promoted by excess hydrolysis reaction, and the proportion of loss of the target polyunsaturated fatty acid increases as concentration factor increases. Thus, at a certain point, the target polyunsaturated fatty acid concentration factor stops increasing, and there is decline in the target polyunsaturated fatty acid concentration. Of course, promotion of hydrolysis may cause a lowering of yield of glycerides. Thus, a limit point occurs in the concentration of a polyunsaturated fatty acid utilizing the hydrolysis by lipase. The added amount of enzyme, reaction time, or the like must be set to obtain a balance between the obtained oil product yield and efficiency of concentration of the target fatty acid.
Optimum temperature of the enzyme reaction is known to depend on the enzyme, and reactions are performed within the temperature range. Although lipase reacts within the temperature range thereof, viscosity of the target oil of the lipase reaction increases at low temperature, and the effectiveness of stirring the oil and enzyme-containing water worsens. Thus, the reaction is normally performed at from 30 to 40° C. For example, when Candida cylindracea-derived lipase is used for concentration of polyunsaturated fatty acid, the reaction temperature used in the working examples of Patent Document 1 (filed in 1982) was room temperature, and thereafter reaction temperatures were set in Patent Documents 2 to 7 (filed in 1988, 1993, 1994, 1995, 1996, and 1999, respectively) to 37, 37, 37, 30, 35, and 35° C., respectively.